Proteomic identification of non-Gal antibody targets after pig-to-primate cardiac xenotransplantation.
BACKGROUND
Experience with non-antigenic galactose alpha1,3 galactose (alphaGal) polymers and development of alphaGal deficient pigs has reduced or eliminated the significance of this antigen in xenograft rejection. Despite these advances, delayed xenograft rejection (DXR) continues to occur most likely due to antibody responses to non-Gal endothelial cell (EC) antigens.


METHODS
To gauge the diversity of the non-Gal antibody response we used antibody derived from CD46 transgenic heterotopic cardiac xenografts performed without T-cell immunosuppression, Group A (n = 4) and Gal knockout (GT-KO) heart transplants under tacrolimus and sirolimus immunosuppression, Group B (n = 8). Non-Gal antibody was measured by flow cytometry and by western blots using GT-KO EC membrane antigens. A nanoLC/MS/MS analysis of proteins recovered from 2D gels was used to identify target antigens.


RESULTS
Group A recipients exhibited a mixed cellular and humoral rejection. Group B recipients mainly exhibited classical DXR. Western blot analysis showed a non-Gal antibody response induced by GT+ and GT-KO hearts to an overlapping set of pig aortic EC membrane antigens. Proteomic analysis identified 14 potential target antigens but failed to define several immunodominant targets.


CONCLUSIONS
These experiments indicate that the non-Gal antibody response is directed to a number of stress response and inflammation related pig EC antigens and a few undefined targets. Further analysis of these antibody specificities using alternative methods is required to more fully define the repertoire of non-Gal antibody responses.
Byrne
Guerard W
GW
Department of Surgery, Mayo Clinic, Rochester, MN 55905, USA. byrne.guerard@mayo.edu
Stalboerger
Paul G
PG
Davila
Eduardo
E
Heppelmann
Carrie J
CJ
Gazi
Mozammel H
MH
McGregor
Hugh C J
HC
LaBreche
Peter T
PT
Davies
William R
WR
Rao
Vinay P
VP
Oi
Keiji
K
Tazelaar
Henry D
HD
Logan
John S
JS
McGregor
Christopher G A
CG
D000818:Animals; D030801:Animals, Genetically Modified; D000910:Antibodies, Heterophile / Q000096:biosynthesis*; D000918:Antibody Specificity; D004187:Disaccharides / Q000172:deficiency / Q000235:genetics / Q000276:immunology; D006084:Graft Rejection / Q000209:etiology / Q000276:immunology / Q000473:pathology; D016027:Heart Transplantation / Q000009:adverse effects* / Q000276:immunology* / Q000473:pathology; D006801:Humans; D048530:Papio anubis; D040901:Proteomics; D013552:Swine; D014183:Transplantation, Heterologous
D000910:Antibodies, Heterophile; D004187:Disaccharides; C055075:galactosyl-(1-3)galactose